PROJECT SUMMARY/ABSTRACT: Diabetes affects ~23 million people in the US alone with the most prevalent form of the disease being type-2 diabetes (T2D), which accounts for ~90%, of diagnosed cases. Obesity, physical inactivity coupled with genetic susceptibility is associated with the rising prevalence of T2D. The risk of cardiovascular disease (CVD) increases 3-fold in diabetes and involves several factors, including hyperglycemia, insulin resistance and dyslipidemia. Peripheral artery disease (PAD) commonly occurs in diabetes and manifests as occlusive arterial disease of the lower extremities. PAD generally points to poor prognosis because it is indicative of wider CVD risk especially involving the cerebrovascular, coronary and renovascular systems. Vascular smooth muscle cell (myocyte) membrane potential is a major regulator of arterial contractility. Diabetes can alter the expression and activity of several ion channels in the vasculature that are associated with intracellular calcium (Ca2+) signaling. Transmembrane protein 16A (TMEM16A, Anoctamin1, ANO1) channels are Ca2+-activated chloride (Cl?) channels that are expressed in arterial myocytes and triggers Cl- efflux, myocyte membrane depolarization and vasoconstriction. Arterial myocyte ion channel gene expression is tightly regulated by signaling mechanisms mediated by several transcription factors. A pathological alteration in these regulatory mechanisms may affect channel expression and induce vascular dysfunction in diabetes. The role of arterial myocyte ANO1 in the development of diabetic vascular dysfunction has not been investigated. This application stems from novel and exciting data of specific signaling mechanisms that regulate Ano1 expression in arterial myocytes to control arterial contractility. Diabetes-induced dysregulation of these signaling pathways leads to increased arterial myocyte ANO1 expression, ANO1 currents and vasoconstriction. The goal of this proposal is to identify key proteins and signaling mechanisms mediating Ano1 expression in resistance arteries and test the therapeutic potential of novel compounds in alleviating diabetes- induced vasoconstriction.